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Publication numberUS6511826 B2
Publication typeGrant
Application numberUS 09/502,783
Publication dateJan 28, 2003
Filing dateFeb 11, 2000
Priority dateJun 6, 1995
Fee statusPaid
Also published asUS6025154, US6759519, US6800729, US7160546, US20020076745, US20020099176, US20020132269, US20030023044, US20050196831
Publication number09502783, 502783, US 6511826 B2, US 6511826B2, US-B2-6511826, US6511826 B2, US6511826B2
InventorsYi Li, Steven M. Ruben
Original AssigneeHuman Genome Sciences, Inc.
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Polynucleotides encoding human G-protein chemokine receptor (CCR5) HDGNR10
US 6511826 B2
Abstract
Human G-protein chemokine receptor polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptides for identifying antagonists and agonists to such polypeptides and methods of using the agonists and antagonists therapeutically to treat conditions related to the underexpression and overexpression of the G-protein chemokine receptor polypeptides, respectively. Also disclosed are diagnostic methods for detecting a mutation in the G-protein chemokine receptor nucleic acid sequences and detecting a level of the soluble form of the receptors in a sample derived from a host.
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Claims(88)
What is claimed is:
1. An isolated polynucleotide comprising a nucleic acid encoding a polypeptide at least 90% identical to the amino acid sequence of SEQ ID NO:2, wherein said polypeptide binds a chemokine which the protein of SEQ ID NO:2 also binds.
2. The polynucleotide of claim 1, which further comprises a heterologous nucleic acid.
3. The polynucleotide of claim 2, wherein said heterologous nucleic acid encodes a heterologous polypeptide.
4. A vector comprising the polynucleotide of claim 1.
5. The vector of claim 4 wherein said polynucleotide is operably linked to a regulatory sequence.
6. A host cell comprising the vector of claim 4.
7. A host cell comprising the vector of claim 5.
8. A host cell comprising the polynucleotide of claim 1 operably linked to a regulatory sequence.
9. A method for producing a G-protein chemokine receptor polypeptide comprising culturing a host cell comprising the polynucleotide of claim 1 and recovering the polypeptide encoded by said polynucleotide.
10. A composition comprising the polynucleotide of claim 1 and a pharmaceutically acceptable carrier.
11. An isolated polynucleotide comprising a nucleic acid encoding a polypeptide at least 95% identical to the amino acid sequence of SEQ ID NO:2, wherein said polypeptide binds a chemokine which the protein of SEQ ID NO:2 also binds.
12. The polynucleotide of claim 11, which further comprises a heterologous nucleic acid.
13. The polynucleotide of claim 12, wherein said heterologous nucleic acid encodes a heterologous polypeptide.
14. A vector comprising the polynucleotide of claim 11.
15. The vector of claim 14 wherein said polynucleotide is operably linked to a regulatory sequence.
16. A host cell comprising the vector of claim 14.
17. A host cell comprising the vector of claim 15.
18. A host cell comprising the polynucleotide of claim 11 operably linked to a regulatory sequence.
19. A method for producing a G-protein chemokine receptor polypeptide comprising culturing a host cell comprising the polynucleotide of claim 11 and recovering the polypeptide encoded by said polynucleotide.
20. A composition comprising the polynucleotide of claim 11 and a pharmaceutically acceptable carrier.
21. An isolated polynucleotide comprising a nucleic acid encoding a polypeptide at least 90% identical to the amino acid sequence encoded by the HDGNR10 coding region in ATCC Deposit No. 97183, wherein said polypeptide binds a chemokine which the protein encoded by the HDGNR10 coding region in ATCC Deposit No. 97183 also binds.
22. The polynucleotide of claim 21, which further comprises a heterologous nucleic acid.
23. The polynucleotide of claim 22, wherein said heterologous nucleic acid encodes a heterologous polypeptide.
24. A vector comprising the polynucleotide of claim 21.
25. The vector of claim 24 wherein said polynucleotide is operably linked to a regulatory sequence.
26. A host cell comprising the vector of claim 24.
27. A host cell comprising the vector of claim 25.
28. A host cell comprising the polynucleotide of claim 21 operably linked to a regulatory sequence.
29. A method for producing a G-protein chemokine receptor polypeptide comprising culturing a host cell comprising the polynucleotide of claim 21 and recovering the polypeptide encoded by said polynucleotide.
30. A composition comprising the polynucleotide of claim 21 and a pharmaceutically acceptable carrier.
31. An isolatcd polynucleotide comprising a nucleic acid encoding a polypeptide at least 95% identical to the amino acid sequence encoded by the HDGNR10 coding region in ATCC Deposit No. 97183, wherein said polypeptide binds a chemokine which the protein encoded by the HDGNR10 coding region in ATCC Deposit No. 97183 also binds.
32. The polynucleotide of claim 31, which further comprises a heterologous nucleic acid.
33. The polynucleotide of claim 32, wherein said heterologous nucleic acid encodes a heterologous polypeptide.
34. A vector comprising the polynucleotide of claim 31.
35. The vector of claim 34 wherein said polynucleotide is operably linked to a regulatory sequence.
36. A host cell comprising the vector of claim 34.
37. A host cell comprising the vector of claim 35.
38. A host cell comprising the polynucleotide of claim 31 operably linked to a regulatory sequence.
39. A method for producing a G-protein chemokine receptor polypeptide comprising culturing a host cell comprising the polynucleotide of claim 31 and recovering the polypeptide encoded by said polynucleotide.
40. A composition comprising the polynucleotide of claim 31 and a pharmaceutically acceptable carrier.
41. An isolated polynucleotide comprising a nucleic acid at least 90% identical to SEQ ID NO:1, wherein said nucleic acid encodes a polypeptide which binds a chemokine which binds the protein of SEQ ID NO:2also binds.
42. The polynucleotide of claim 41, which further comprises a heterologous nucleic acid.
43. A vector comprising the polynucleotide of claim 41.
44. The vector of claim 43 wherein said polynucleotide is operably linked to a regulatory sequence.
45. A host cell comprising the vector of claim 43.
46. A host cell comprising the vector of claim 44.
47. A host cell comprising the polynucleotide of claim 41 operably linked to a regulatory sequence.
48. A composition comprising the polynucleotide of claim 41 and a pharmaceutically acceptable carrier.
49. An isolated polynucleotide comprising a nucleic acid at least 95% identical to SEQ ID NO:1, wherein said nucleic acid encodes a polypeptide which binds a chemokine which the protein of SEQ ID NO:2 also binds.
50. The polynucleotide of claim 49, which further comprises a heterologous nucleic acid.
51. A vector comprising the polynucleotide of claim 49.
52. The vector of claim 51 wherein said polynucleotide is operably linked to a regulatory sequence.
53. A host cell comprising the vector of claim 51.
54. A host cell comprising the vector of claim 52.
55. A host cell comprising the polynucleotide of claim 49 operably linked to a regulatory sequence.
56. A composition comprising the polynucleotide of claim 49 and a pharmaceutically acceptable carrier.
57. An isolated polynucleotide comprising a nucleic acid at least 90% identical to the HDGNR10 coding region in ATCC Deposit No. 97183, wherein said nucleic acid encodes a polypeptide which binds a chemokine which the protein encoded by the HDGNR10 coding region in ATCC Deposit No. 97183 also binds.
58. The polynucleotide of claim 57, which further comprises a heterologous nucleic acid.
59. A vector comprising the polynucleotide of claim 57.
60. The vector of claim 59 wherein said polynucleotide is operably linked to a regulatory sequence.
61. A host cell comprising the vector of claim 59.
62. A host cell comprising the vector of claim 60.
63. A host cell comprising the polynucleotide of claim 57 operably linked to a regulatory sequence.
64. A composition comprising the polynucleotide of claim 57 and a pharmaceutically acceptable carrier.
65. An isolated polynucleotide comprising a nucleic acid at least 95% identical to the HDGNR10 coding region in ATCC Deposit No. 97183, wherein said nucleic acid encodes a polypeptide which binds a chemokine which the protein encoded by the HDGNR10 coding region in ATCC Deposit No. 97183 also binds.
66. The polynucleotide of claim 65, which further comprises a heterologous nucleic acid.
67. A vector comprising the polynucleotide of claim 65.
68. The vector of claim 67 wherein said polynucleotide is operably linked to a regulatory sequence.
69. A host cell comprising the vector of claim 67.
70. A host cell comprising the vector of claim 68.
71. A host cell comprising the polynucleotide of claim 65 operably linked to a regulatory sequence.
72. A composition comprising the polynucleotide of claim 65 and a pharmaceutically acceptable carrier.
73. The full complement of the polynucleotide of claim 1.
74. The full complement of the polynucleotide of claim 11.
75. The full complement of the polynucleotide of claim 21.
76. The full complement of the polynucleotide of claim 31.
77. The full complement of the polynucleotide of claim 41.
78. The full complement of the polynucleotide of claim 49.
79. The full complement of the polynucleotide of claim 57.
80. The full complement of the polynucleotide of claim 65.
81. The polynucleotide of claim 42, wherein said heterologous nucleic acid encodes a heterologous polypeptide.
82. A method for producing a G-protein chemokine receptor polypeptide comprising culturing a host cell comprising the polynucleotide of claim 41 and recovering the polypeptide encoded by said polynucleotide.
83. The polynucleotide of claim 50, wherein said heterologous nucleic acid encodes a hererologous polypeptide.
84. A method for producing a G-protein chemokine receptor polypeptide comprising culturing a host cell comprising the polynucleotide of claim 49 and recovering the polypeptide encoded by said polynucleotide.
85. The polynucleotide of claim 58, wherein said heterologous nucleic acid encodes a heterologous polypeptide.
86. A method for producing a G-protein chemokine receptor polypeptide comprising culturing a host cell comprising the polynucleotide of claim 57 and recovering the polypeptide encoded by said polynucleotide.
87. The polynucleotide of claim 66, wherein said heterologous nucleic acid encodes a heterologous polypeptide.
88. A method for producing a G-protein chemokine receptor polypeptide comprising culturing a host cell comprising the polynucleotide of claim 65 and recovering the polypeptide encoded by said polynucleotide.
Description

The present application is a continution of U.S. patent application No. 08/466,343, filed Jun. 6, 1995, Pat. No. 6,025,154, which disclosure is herein incorporated be reference.

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a human 7-transmembrane receptor which has been putatively identified as a chemokine receptor, sometimes hereinafter referred to as “G-Protein Chemokine Receptor” or “HDGNR10”. The invention also relates to inhibiting the action of such polypeptides.

It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 351:353-354 (1991)). Herein these proteins are referred to as proteins participating in pathways with G-proteins or PPG proteins. Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B. K., et al., PNAS, 84:46-50 (1987); Kobilka, B. K., et al., Science, 238:650-656 (1987); Bunzow, J. R., et al., Nature, 336:783-787 (1988)), G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M. I., et al., Science, 252:802-8 (1991)).

For example, in one form of signal transduction, the effect of hormone binding is activation of an enzyme, adenylate cyclase, inside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide GTP, and GTP also influences hormone binding. A G-protein connects the hormone receptors to adenylate cyclase. G-protein was shown to exchange GTP for bound GDP when activated by hormone receptors. The GTP-carrying form then binds to an activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns the G-protein to its basal, inactive form. Thus, the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.

The membrane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane α-helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.

G-protein coupled receptors have been characterized as including these seven conserved hydrophobic stretches of about 20 -to 30 amino acids, connecting at least eight divergent hydrophilic loops. The G-protein family of coupled receptors includes dopamine receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders. Other examples of members of this family include calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-l receptor and rhodopsins, odorant, cytomegalovirus receptors, etc.

G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc., Rev., 10:317-331 (1989)). Different G-protein α-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protein coupled receptors have been identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors. G-protein coupled receptors are found in numerous sites within a mammalian host.

Chemokines, also referred to as intercrine cytokines, are a subfamily of structurally and functionally related cytokines. These molecules are 8-10 kd in size. In general, chemokines exhibit 20% to 75% homology at the amino acid level and are characterized by four conserved cysteine residues that form two disulfide bonds. Based on the arrangement of the first two cysteine residues, chemokines have been classified into two subfamilies, alpha and beta. In the alpha subfamily, the first two cysteines are separated by one amino acid and hence are referred to as the “C-X-C” subfamily. In the beta subfamily, the two cysteines are in an adjacent position and are, therefore, referred to as the “C-C” subfamily. Thus far, at least nine different members of this family have been identified in humans.

The intercrine cytokines exhibit a wide variety of functions. A hallmark feature is their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutrophils, T lymphocytes, basophils and fibroblasts. Many chemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. These activities include stimulation of histamine release, lysosomal enzyme and leukotriene release, increased adherence of target immune cells to endothelial cells, enhanced binding of complement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to their involvement in inflammation, certain chemokines have been shown to exhibit other activities. For example, macrophage inflammatory protein 1 (MIP-1) is able to suppress hematopoietic stem cell proliferation, platelet factor-4 (PF-4) is a potent inhibitor of endothelial cell growth, Interleukin-8 (IL-8) promotes proliferation of keratinocytes, and GRO is an autocrine growth factor for melanoma cells.

In light of the diverse biological activities, it is not surprising that chemokines have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there are provided novel mature receptor polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The receptor polypeptides of the present invention are of human origin.

In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the receptor polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA as well as antisense analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.

In accordance with a further aspect of the present invention, there are provided processes for producing such receptor polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing nucleic acid sequences encoding the receptor polypeptides of the present invention, under conditions promoting expression of said polypeptides and subsequent recovery of said polypeptides.

In accordance with yet a further aspect of the present invention, there are provided antibodies against such receptor polypeptides.

In accordance with another aspect of the present invention there are provided methods of screening for compounds which bind to and activate or inhibit activation of the receptor polypeptides of the present invention.

In accordance with still another embodiment of the present invention there are provided processes of administering compounds to a host which bind to and activate the receptor polypeptide of the present invention which are useful in stimulating haematopoiesis, wound healing, coagulation, angiogenesis, to treat solid tumors, chronic infections, leukemia, T-cell mediated auto-immune diseases, parasitic infections, psoriasis, and to stimulate growth factor activity.

In accordance with another aspect of the present invention there is provided a method of administering the receptor polypeptides of the present invention via gene therapy to treat conditions related to underexpression of the polypeptides or underexpression of a ligand for the receptor polypeptide.

In accordance with still another embodiment of the present invention there are provided processes of administering compounds to a host which bind to and inhibit activation of the receptor polypeptides of the present invention which are useful in the prevention and/or treatment of allergy, atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation, histamine and IgE-mediated allergic reactions, prostaglandin-independent fever, bone marrow failure, silicosis, sarcoidosis, rheumatoid arthritis, shock and hyper-eosinophilic syndrome.

In accordance with yet another aspect of the present invention, there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the polynucleotide sequences of the present invention.

In accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences encoding such polypeptides and for detecting an altered level of the soluble form of the receptor polypeptides.

In accordance with yet a further aspect of the present invention, there are provided processes for utilizing such receptor polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

FIGS. 1A-1C show the DNA sequence and the corresponding deduced amino acid sequence of the G-protein coupled receptor of the present invention. The standard one-letter abbreviation for amino acids is used. Sequencing was performed using a 373 Automated DNA sequencer (Applied Biosystems, Inc.).

FIG. 2 illustrates an amino acid alignment, utilizing the standard one-letter abbreviation for amino acids, of the G-protein chemokine receptor of the present invention (a portion of SEQ ID NO:2) and a comparative portion (SEQ ID NO:9) of the amino acid sequence for the human MCP-1 receptor.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of FIGS. 1A-1C (SEQ ID NO:2) or for the mature polypeptide encoded by the HDGNR10 coding region of the clone deposited as ATCC Deposit No. 97183 on Jun. 1, 1995 at the American Type Culture Collection, Patent Depository, 10801 Unversity Boulevard, Manassas, Va. 20110-2209.

The polynucleotide of this invention was discovered in a human genomic DNA. It is structurally related to the G protein-coupled receptor family. It contains an open reading frame encoding a protein of 352 amino acid residues. The protein exhibits the highest degree of homology to a human MCP-1 receptor (SEQ ID NO:9) with 70.1% identity and 82.9% similarity over a 347 amino acid stretch.

The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in FIGS. 1A-1C (SEQ ID NO:1) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of FIGS. 1A-1C (SEQ ID NO:1) or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of FIGS. 1A-1C or for the mature polypeptide encoded by the deposited clone may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a transmembrane (TM) or intra-cellular domain; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5′ and/or 3′ of the coding sequence for the mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of FIGS. 1A-1C or the polypeptide encoded by the deposited clone. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in FIG. 1 (SEQ ID NO:2) or the same mature polypeptide encoded by the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of FIG. 1 (SEQ ID NO:2) or the polypeptide encoded by the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in FIG. 1 (SEQ ID NO:1) or of the coding sequence of the deposited clone. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.

The polynucleotides may also encode for a soluble form of the G-protein chemokine receptor polypeptide which is the extracellular portion of the polypeptide which has been cleaved from the TM and intracellular domain of the full-length polypeptide of the present invention.

The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexahistidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term “stringent conditions” means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the DNA of FIG. 1 (SEQ ID NO:1) or the deposited clones.

Alternatively, the polynucleotide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO:1, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotides.

The deposit(s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, or sell the deposited materials, and no such license is hereby granted.

The present invention further relates to a G-protein chemokine receptor polypeptide which has the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2) or which has the amino acid sequence encoded by the HDGNR10 coding region of the deposited clone, as well as fragments, analogs and derivatives of such polypeptide.

The terms “fragment,” “derivative” and “analog” when referring to the polypeptide of FIG. 1 or that encoded by the deposited clone, means a polypeptide which either retains substantially the same biological function or activity as such polypeptide, i.e. functions as a G-protein chemokine receptor, or retains the ability to bind the ligand or the receptor even though the polypeptide does not function as a G-protein chemokine receptor, for example, a soluble form of the receptor. An analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 (SEQ ID NO:2) or that encoded by the deposited clone may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide for purification of the polypeptide or (v) one in which a fragment of the polypeptide is soluble, i.e. not membrane bound, yet still binds ligands to the membrane bound receptor. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70% similarity (preferably a 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably a 90% similarity (more preferably a 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably a 95% similarity (still more preferably a 90% identity) to the polypeptide of SEQ ID NO:2 and to portions of such polypeptide with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.

The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region “leader and trailer” as well as intervening sequences (introns) between individual coding segments (exons).

The term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host. However, any other vector may be used as long as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenovirus; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, PBPV, PMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level. of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

The G-protein chemokine receptor polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human disease.

The G-protein chemokine receptors of the present invention may be employed in a process for screening for compounds which activate (agonists) or inhibit activation (antagonists) of the receptor polypeptide of the present invention

In general, such screening procedures involve providing appropriate cells which express the receptor polypeptide of the present invention on the surface thereof. Such cells include cells from mammals, yeast, drosophila or E. Coli. In particular, a polynucleotide encoding the receptor of the present invention is employed to transfect cells to thereby express the G-protein chemokine receptor. The expressed receptor is then contacted with a test compound to observe binding, stimulation or inhibition of a functional response.

One such screening procedure involves the use of melanophores which are transfected to express the G-protein chemokine receptor of the present invention. Such a screening technique is described in PCT WO 92/01810 published Feb. 6, 1992.

Thus, for example, such assay may be employed for screening for a compound which inhibits activation of the receptor polypeptide of the present invention by contacting the melanophore cells which encode the receptor with both the receptor ligand and a compound to be screened. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, i.e., inhibits activation of the receptor.

The screen may be employed for determining a compound which activates the receptor by contacting such cells with compounds to be screened and determining whether such compound generates a signal, i.e., activates the receptor.

Other screening techniques include the use of cells which express the G-protein chemokine receptor (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science,volume 246, pages 181-296 (October 1989). For example, compounds may be contacted with a cell which expresses the receptor polypeptide of the present invention and a second messenger response, e.g. signal transduction or pH changes, may be measured to determine whether the potential compound activates or inhibits the receptor.

Another such screening technique involves introducing RNA encoding the G-protein chemokine receptor into Xenopus oocytes to transiently express the receptor. The receptor oocytes may then be contacted with the receptor ligand and a compound to be screened, followed by detection of inhibition or activation of a calcium signal in the case of screening for compounds which are thought to inhibit activation of the receptor.

Another screening technique involves expressing the G-protein chemokine receptor in which the receptor is linked to a phospholipase C or D. As representative examples of such cells, there may be mentioned endothelial cells, smooth muscle cells, embryonic kidney cells, etc. The screening may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase second signal.

Another method involves screening for compounds which inhibit activation of the receptor polypeptide of the present invention antagonists by determining inhibition of binding of labeled ligand to cells which have the receptor on the surface thereof. Such a method involves transfecting a eukaryotic cell with DNA encoding the G-protein chemokine receptor such that the cell expresses the receptor on its surface and contacting the cell with a compound in the presence of a labeled form of a known ligand. The ligand can be labeled, e.g., by radioactivity. The amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. If the compound binds to the receptor as determined by a reduction of labeled ligand which binds to the receptors, the binding of labeled ligand to the receptor is inhibited.

An antibody against the G-protein chemokine receptor of the invention may agonize or may antagonize the activity of the receptor.

An antibody may antagonize a G-protein chemokine receptor of the present invention, or in some cases an oligopeptide, which bind to the G-protein chemokine receptor but does not elicit a second messenger response such that the activity of the G-protein chemokine receptors is prevented. Antibodies include anti-idiotypic antibodies which recognize unique determinants generally associated with the antigen-binding site of an antibody. Potential antagonist compounds also include proteins which are closely related to the ligand of the G-protein chemokine receptors, i.e. a fragment of the ligand, which have lost biological function and when binding to the G-protein chemokine receptor elicit no response.

An antisense construct prepared through the use of antisense technology, may be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5′ coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of G-protein chemokine receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of mRNA molecules into G-protein coupled receptor (antisense—Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of G-protein chemokine receptor.

A small molecule which binds to the G-protein chemokine receptor, making it inaccessible to ligands such that normal biological activity is prevented, for example small peptides or peptide-like molecules, may also be used to inhibit activation of the receptor polypeptide of the present invention.

A soluble form of the G-protein chemokine receptor, e.g. a fragment of the receptors, may be used to inhibit activation of the receptor by binding to the ligand to a polypeptide of the present invention and preventing the ligand from interacting with membrane bound G-protein chemokine receptors.

The compounds which bind to and activate the G-protein chemokine receptors of the present invention may be employed to stimulate haematopoiesis, wound healing, coagulation, angiogenesis, to treat solid tumors, chronic infections, leukemia, T-cell mediated auto-immune diseases, parasitic infections, psoriasis, and to stimulate growth factor activity.

The compounds which bind to and inhibit the G-protein chemokine receptors of the present invention may be employed to treat allergy, atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation, histamine and IgE-mediated allergic reactions, prostaglandin-independent fever, bone marrow failure, silicosis, sarcoidosis, rheumatoid arthritis, shock and hyper-eosinophilic syndrome.

The compounds may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the compounds of the present invention may be employed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenient manner such as by the topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, the pharmaceutical compositions will be administered in an amount of at least about 10 μg/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 μg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.

The G-protein chemokine receptor polypeptides and antagonists or agonists which are polypeptides, may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as “gene therapy.”

Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.

Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and β-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the β-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controls the genes encoding the polypeptides.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAM12, and DAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

The present invention also provides a method for determining whether a ligand not known to be capable of binding to a G-protein chemokine receptor can bind to such receptor which comprises contacting a mammalian cell which expresses a G-protein chemokine receptor with the ligand under conditions permitting binding of ligands to the G-protein chemokine receptor, detecting the presence of a ligand which binds to the receptor and thereby determining whether the ligand binds to the G-protein chemokine receptor. The systems hereinabove described for determining agonists and/or antagonists may also be employed for determining ligands which bind to the receptor.

This invention also provides a method of detecting expression of a G-protein chemokine receptor polypeptide of the present invention on the surface of a cell by detecting the presence of mRNA coding for the receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule of at least 10 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding the receptor under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the receptor by the cell.

The present invention also provides a method for identifying receptors related to the receptor polypeptides of the present invention. These related receptors may be identified by homology to a G-protein chemokine receptor polypeptide of the present invention, by low stringency cross hybridization, or by identifying receptors that interact with related natural or synthetic ligands and or elicit similar behaviors after genetic or pharmacological blockade of the chemokine receptor polypeptides of the present invention.

Fragments of the genes may be used as a hybridization probe for a cDNA library to isolate other genes which have a high sequence similarity to the genes of the present invention, or which have similar biological activity. Probes of this type are at least 20 bases, preferably at least 30 bases and most preferably at least 50 bases or more. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene of the present invention including regulatory and promoter regions, exons and introns. An example of a screen of this type comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the genes of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

The present invention also contemplates the use of the genes of the present invention as a diagnostic, for example, some diseases result from inherited defective genes. These genes can be detected by comparing the sequences of the defective gene with that of a normal one. Subsequently, one can verify that a “mutants” gene is associated with abnormal receptor activity. In addition, one can insert mutant receptor genes into a suitable vector for expression in a functional assay system (e.g., colorimetric assay, expression on MacConkey plates, complementation experiments, in a receptor deficient strain of HEK293 cells) as yet another means to verify or identify mutations. Once “mutant” genes have been identified, one can then screen population for carriers of the “mutant” receptor gene.

Individuals carrying mutations in the gene of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids used for diagnosis may be obtained from a patient's cells, including but not limited to such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki, et al., Nature, 324:163-166 1986) prior to analysis. RNA or cDNA may also be used for the same purpose. As an example, PCR primers complimentary to the nucleic acid of the instant invention can be used to identify and analyze mutations in the gene of the present invention. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radio labeled RNA of the invention or alternatively, radio labeled antisense DNA sequences of the invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures. Such a diagnostic would be particularly useful for prenatal or even neonatal testing.

Sequence differences between the reference gene and “mutants” may be revealed by the direct DNA sequencing method. In addition, cloned DNA segments may be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequence primer is used with double stranded PCR product or a single stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radio labeled nucleotide or by an automatic sequencing procedure with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved by detection of alterations in the electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Sequences changes at specific locations may also be revealed by nucleus protection assays, such RNase and S1 protection or the chemical cleavage method (e.g. Cotton, et al., PNAS, USA, 85:4397-4401 1985).

In addition, some diseases are a result of, or are characterized by changes in gene expression which can be detected by changes in the mRNA. Alternatively, the genes of the present invention can be used as a reference to identify individuals expressing a decrease of functions associated with receptors of this type.

The present invention also relates to a diagnostic assay for detecting altered levels of soluble forms of the G-protein chemokine receptor polypeptides of the present invention in various tissues. Assays used to detect levels of the soluble receptor polypeptides in a sample derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive-binding assays, Western blot analysis and preferably as ELISA assay.

An ELISA assay initially comprises preparing an antibody specific to antigens of the G-protein chemokine receptor polypeptides, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or in this example a horseradish peroxidase enzyme. A sample is now removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any G-protein chemokine receptor proteins attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to G-protein chemokine receptor proteins. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of G-protein chemokine receptor proteins present in a given volume of patient sample when compared against a standard curve.

The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the DNA is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60 bases. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.

With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., (1985), pp. 77-96).

Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of this invention.

The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.

In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37° C. are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 8:4057 (1980).

“Oligonucleotides” refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5′ phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase (“ligase”) per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.

Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

EXAMPLE 1 Bacterial Expression and Purification of HDGNR10

The DNA sequence encoding for HDGNR10, ATCC No. 97183 is initially amplified using PCR oligonucleotide primers corresponding to the 5′ and sequences of the processed HDGNR10 protein (minus the signal peptide sequence) and the vector sequences 3′ to the HDGNR10 gene. Additional nucleotides corresponding to HDGNR10 were added to the 5′ and 3′ sequences respectively. The 5′ oligonucleotide primer has the sequence 5′ CGGAATRCCTCCATGGATTATCAAGTGTCA 3′ (SEQ ID NO:3) contains an EcoRI restriction enzyme site followed by 18 nucleotides of HDGNR10 coding sequence starting from the presumed terminal amino acid of the processed protein codon. The 3′ sequence 5′ CGGAAGCTTCGTCACAAGCCCACAGATAT 3′ (SEQ ID NO:4) contains complementary sequences to a HindIII site and is followed by 18 nucleotides of HDGNR10 coding sequence. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, Calif., 91311). pQE-9 encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites. pQE-9 was then digested with EcoRI and HindIII. The amplified sequences were ligated into pQE-9 and were inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture was then used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989). M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kanr). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysis. Clones containing the desired constructs were grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells were grown to an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) was then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression. Cells were grown an extra 3 to 4 hours. Cells were then harvested by centrifugation. The cell pellet was solubilized in the chaotropic agent 6 Molar Guanidine HCl. After clarification, solubilized HDGNR10 was purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag. Hochuli, E. et al., J. Chromatography 411:177-184 (1984). HDGNR10 was eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized). After incubation in this solution for 12 hours the protein was dialyzed to 10 mmolar sodium phosphate.

EXAMPLE 2 Expression of Recombinant HDGNR10 in COS cells

The expression of plasmid, HDGNR10 HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA fragment encoding the entire HDGNR10 precursor and a HA tag fused in frame to its 3′ end was cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, et al. Cell 37, 767(1984)). The infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding for HDGNR10, ATCC #97183, was constructed by PCR using two primers: the 5′ primer 5′ GTCCAAGCTTGCCACCATGGATTATCAAGTGTCA 3′ (SEQ ID NO:5) and contains a HindIII site followed by 18 nucleotides of HDGNR10 coding sequence starting from the initiation codon; the 3′ sequence 5′ CTAGCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGGGTAGCACAAGCCCACAGATATTC 3′ (SEQ ID NO:6) contains complementary sequences to an XhoI site, translation stop codon, HA tag and the last 18 nucleotides of the HDGNR10 coding sequence (not including the stop codon). Therefore, the PCR product contains a HindIII site HDGNR10 coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an XhoI site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were digested with HindIII and XhoI restriction enzyme and ligated. The ligation mixture was transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037) the transformed culture was plated on ampicillin media plates and resistant colonies were selected. Plasmid DNA was isolated from transformants and examined by restriction analysis for the presence of the correct fragment. For expression of the recombinant HDGNR10, COS cells were transfected with the expression vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). The expression of the HDGNR10 HA protein was detected by radiolabelling and immunoprecipitation method. (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours with 35S-cysteine two days post transfection. Culture media were then collected and cells were lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5). (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culture media were precipitated with a HA specific monoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGE gels.

EXAMPLE 3 Cloning and Expression of HDGNR10 Using the Baculovirus Expression System

The DNA sequence encoding the full length HDGNR10 protein, ATCC 97183, was amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ sequences of the gene:

The 5′ primer has the sequence 5′ CGGGATCCCTCCATGGATTATCAAGTGTCA 3′ (SEQ ID NO:7) and contains a BamHI restriction enzyme site followed by 4 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol. 196: 947-950 ,(1987)), and just behind the first 18 nucleotides of the HDGNR10 gene (the initiation codon for translation is “ATG”).

The 3′ primer has the sequence 5′ CGGGATCCCGCTCACAAGCCCACAGATAT 3′ (SEQ ID NO:8) and contains the cleavage site for the restriction endonuclease BamHI and 18 nucleotides complementary to the 3′ non-translated sequence of the HDGNR10 gene. The amplified sequences were isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment was then digested with the endonuclease BamHI and purified as described above. This fragment is designated F2.

The vector PRG1 (modification of pVL941 vector, discussed below) is used for the expression of the HDGNR10 protein using the baculovirus expression system (for review see: Summers, M. D. and Smith, G. E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555). This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction endonuclease BamHI. The polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation. For an easy selection of recombinant viruses the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recombination of co-transfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology, 170:31-39).

The plasmid was digested with the restriction enzyme BamHI and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The DNA was then isolated from a 1% agarose gel as described above. This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase. E.coli HB101 cells were then transformed and bacteria identified that contained the plasmid (pBacHDGNR10) with the HDGNR10 gene using the enzyme BamHI. The sequence of the cloned fragment was confirmed by DNA sequencing.

5 μg of the plasmid pBacHDGNR10 were co-transfected with 1.0 μg of a commercially available linearized baculovirus (“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.) using the lipofection method (Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacHDGNR10 were mixed in a sterile well of a microtiter plate containing 50 μl of serum free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace's medium were added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture was added drop wise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate was rocked back and forth to mix the newly added solution. The plate was then incubated for 5 hours at 27° C. After 5 hours the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum was added. The plate was put back into an incubator and cultivation continued at 27° C. for four days.

After four days the supernatant was collected and a plaque assay performed similar as described by Summers and Smith (supra). As a modification an agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) was used which allows an easy isolation of blue stained plaques. (A detailed description of a “plaque assay” can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the viruses were added to the cells, blue stained plaques were picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses was then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar was removed by a brief centrifugation and the supernatant containing the recombinant baculoviruses was used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes were harvested and then stored at 4° C.

Sf 9 cells were grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells were infected with the recombinant baculovirus V-HDGNR10 at a multiplicity of infection (MOI) of 2. Six hours later the medium was removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 μCi of 35S-methionine and 5 μCi 35S cysteine (Amersham) were added. The cells were further incubated for 16 hours before they were harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.

EXAMPLE 4 Expression via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added. This is then incubated at 37° C. for approximately one week. At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.

The DNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5′ and 3′ end sequences respectively. The 5′ primer contains an EcoRI site and the 3′ primer contains a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is used to transform bacteria HB101, which are then plated onto agar-containing kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his.

The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product.

Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.

                  
#             SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS: 9
<210> SEQ ID NO 1
<211> LENGTH: 1414
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (259)..(1314)
<400> SEQUENCE: 1
gtgagatggt gctttcatga attcccccaa caagagccaa gctctccatc ta
#gtggacag     60
ggaagctagc agcaaacctt cccttcacta cgaaacttca ttgcttggcc ca
#aaagagag    120
ttaattcaat gtagacatct atgtaggcaa ttaaaaacct attgatgtat aa
#aacagttt    180
gcattcatgg agggcaacta aatacattct aggactttat aaaagatcac tt
#tttattta    240
tgcacagggt ggaacaag atg gat tat caa gtg tca agt 
#cca atc tat gac      291
                  
#  Met Asp Tyr Gln Val Ser Ser Pro Ile 
#Tyr Asp
                  
#  1               5 
#                  
#10
atc aat tat tat aca tcg gag ccc tgc caa aa
#a atc aat gtg aag caa      339
Ile Asn Tyr Tyr Thr Ser Glu Pro Cys Gln Ly
#s Ile Asn Val Lys Gln
            15      
#            20      
#            25
atc gca gcc cgc ctc ctg cct ccg ctc tac tc
#a ctg gtg ttc atc ttt      387
Ile Ala Ala Arg Leu Leu Pro Pro Leu Tyr Se
#r Leu Val Phe Ile Phe
        30          
#        35          
#        40
ggt ttt gtg ggc aac atg ctg gtc atc ctc at
#c ctg ata aac tgc aaa      435
Gly Phe Val Gly Asn Met Leu Val Ile Leu Il
#e Leu Ile Asn Cys Lys
    45              
#    50              
#    55
agg ctg aag agc atg act gac atc tac ctg ct
#c aac ctg gcc atc tct      483
Arg Leu Lys Ser Met Thr Asp Ile Tyr Leu Le
#u Asn Leu Ala Ile Ser
60                  
#65                  
#70                  
#75
gac ctg ttt ttc ctt ctt act gtc ccc ttc tg
#g gct cac tat gct gcc      531
Asp Leu Phe Phe Leu Leu Thr Val Pro Phe Tr
#p Ala His Tyr Ala Ala
                80  
#                85  
#                90
gcc cag tgg gac ttt gga aat aca atg tgt ca
#a ctc ttg aca ggg ctc      579
Ala Gln Trp Asp Phe Gly Asn Thr Met Cys Gl
#n Leu Leu Thr Gly Leu
            95      
#            100     
#            105
tat ttt ata ggc ttc ttc tct gga atc ttc tt
#c atc atc ctc ctg aca      627
Tyr Phe Ile Gly Phe Phe Ser Gly Ile Phe Ph
#e Ile Ile Leu Leu Thr
        110          
#       115          
#       120
atc gat agg tac ctg gct gtc gtc cat gct gt
#g ttt gct tta aaa gcc      675
Ile Asp Arg Tyr Leu Ala Val Val His Ala Va
#l Phe Ala Leu Lys Ala
    125              
#   130              
#   135
agg acg gtc acc ttt ggg gtg gtg aca agt gt
#g atc act tgg gtg gtg      723
Arg Thr Val Thr Phe Gly Val Val Thr Ser Va
#l Ile Thr Trp Val Val
140                 1
#45                 1
#50                 1
#55
gct gtg ttt gcg tct ctc cca gga atc atc tt
#t acc aga tct caa aaa      771
Ala Val Phe Ala Ser Leu Pro Gly Ile Ile Ph
#e Thr Arg Ser Gln Lys
                160  
#               165  
#               170
gaa ggt ctt cat tac acc tgc agc tct cat tt
#t cca tac agt cag tat      819
Glu Gly Leu His Tyr Thr Cys Ser Ser His Ph
#e Pro Tyr Ser Gln Tyr
            175      
#           180      
#           185
caa ttc tgg aag aat ttc cag aca tta aag at
#a gtc atc ttg ggg ctg      867
Gln Phe Trp Lys Asn Phe Gln Thr Leu Lys Il
#e Val Ile Leu Gly Leu
        190          
#       195          
#       200
gtc ctg ccg ctg ctt gtc atg gtc atc tgc ta
#c tcg gga atc cta aaa      915
Val Leu Pro Leu Leu Val Met Val Ile Cys Ty
#r Ser Gly Ile Leu Lys
    205              
#   210              
#   215
act ctg ctt cgg tgt cga aat gag aag aag ag
#g cac agg gct gtg agg      963
Thr Leu Leu Arg Cys Arg Asn Glu Lys Lys Ar
#g His Arg Ala Val Arg
220                 2
#25                 2
#30                 2
#35
ctt atc ttc acc atc atg att gtt tat ttt ct
#c ttc tgg gct ccc tac     1011
Leu Ile Phe Thr Ile Met Ile Val Tyr Phe Le
#u Phe Trp Ala Pro Tyr
                240  
#               245  
#               250
aac att gtc ctt ctc ctg aac acc ttc cag ga
#a ttc ttt ggc ctg aat     1059
Asn Ile Val Leu Leu Leu Asn Thr Phe Gln Gl
#u Phe Phe Gly Leu Asn
            255      
#           260      
#           265
aat tgc agt agc tct aac agg ttg gac caa gc
#t atg cag gtg aca gag     1107
Asn Cys Ser Ser Ser Asn Arg Leu Asp Gln Al
#a Met Gln Val Thr Glu
        270          
#       275          
#       280
act ctt ggg atg acg cac tgc tgc atc aac cc
#c atc atc tat gcc ttt     1155
Thr Leu Gly Met Thr His Cys Cys Ile Asn Pr
#o Ile Ile Tyr Ala Phe
    285              
#   290              
#   295
gtc ggg gag aag ttc aga aac tac ctc tta gt
#c ttc ttc caa aag cac     1203
Val Gly Glu Lys Phe Arg Asn Tyr Leu Leu Va
#l Phe Phe Gln Lys His
300                 3
#05                 3
#10                 3
#15
att gcc aaa cgc ttc tgc aaa tgc tgt tct at
#t ttc cag caa gag gct     1251
Ile Ala Lys Arg Phe Cys Lys Cys Cys Ser Il
#e Phe Gln Gln Glu Ala
                320  
#               325  
#               330
ccc gag cga gca agc tca gtt tac acc cga tc
#c act gag gag cag gaa     1299
Pro Glu Arg Ala Ser Ser Val Tyr Thr Arg Se
#r Thr Glu Glu Gln Glu
            335      
#           340      
#           345
ata tct gtg ggc ttg tgacacggac tcaagtgggc tggtgaccc
#a gtcagagttg     1354
Ile Ser Val Gly Leu
        350
tgcacatggc ttagttttca tacacagcct gggctggggg tggggtggaa ga
#ggtctttt   1414
<210> SEQ ID NO 2
<211> LENGTH: 352
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2
Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr As
#p Ile Asn Tyr Tyr Thr
1               5   
#                10  
#                15
Ser Glu Pro Cys Gln Lys Ile Asn Val Lys Gl
#n Ile Ala Ala Arg Leu
            20      
#            25      
#            30
Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Ph
#e Gly Phe Val Gly Asn
        35          
#        40          
#        45
Met Leu Val Ile Leu Ile Leu Ile Asn Cys Ly
#s Arg Leu Lys Ser Met
    50              
#    55              
#    60
Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Se
#r Asp Leu Phe Phe Leu
65                  
#70                  
#75                  
#80
Leu Thr Val Pro Phe Trp Ala His Tyr Ala Al
#a Ala Gln Trp Asp Phe
                85  
#                90  
#                95
Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Le
#u Tyr Phe Ile Gly Phe
            100      
#           105      
#           110
Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Th
#r Ile Asp Arg Tyr Leu
        115          
#       120          
#       125
Ala Val Val His Ala Val Phe Ala Leu Lys Al
#a Arg Thr Val Thr Phe
    130              
#   135              
#   140
Gly Val Val Thr Ser Val Ile Thr Trp Val Va
#l Ala Val Phe Ala Ser
145                 1
#50                 1
#55                 1
#60
Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Ly
#s Glu Gly Leu His Tyr
                165  
#               170  
#               175
Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Ty
#r Gln Phe Trp Lys Asn
            180      
#           185      
#           190
Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Le
#u Val Leu Pro Leu Leu
        195          
#       200          
#       205
Val Met Val Ile Cys Tyr Ser Gly Ile Leu Ly
#s Thr Leu Leu Arg Cys
    210              
#   215              
#   220
Arg Asn Glu Lys Lys Arg His Arg Ala Val Ar
#g Leu Ile Phe Thr Ile
225                 2
#30                 2
#35                 2
#40
Met Ile Val Tyr Phe Leu Phe Trp Ala Pro Ty
#r Asn Ile Val Leu Leu
                245  
#               250  
#               255
Leu Asn Thr Phe Gln Glu Phe Phe Gly Leu As
#n Asn Cys Ser Ser Ser
            260      
#           265      
#           270
Asn Arg Leu Asp Gln Ala Met Gln Val Thr Gl
#u Thr Leu Gly Met Thr
        275          
#       280          
#       285
His Cys Cys Ile Asn Pro Ile Ile Tyr Ala Ph
#e Val Gly Glu Lys Phe
    290              
#   295              
#   300
Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys Hi
#s Ile Ala Lys Arg Phe
305                 3
#10                 3
#15                 3
#20
Cys Lys Cys Cys Ser Ile Phe Gln Gln Glu Al
#a Pro Glu Arg Ala Ser
                325  
#               330  
#               335
Ser Val Tyr Thr Arg Ser Thr Glu Glu Gln Gl
#u Ile Ser Val Gly Leu
            340      
#           345      
#           350
<210> SEQ ID NO 3
<211> LENGTH: 30
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide
<400> SEQUENCE: 3
cggaattcct ccatggatta tcaagtgtca         
#                  
#           30
<210> SEQ ID NO 4
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide
<400> SEQUENCE: 4
cggaagcttc gtcacaagcc cacagatat         
#                  
#            29
<210> SEQ ID NO 5
<211> LENGTH: 34
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide
<400> SEQUENCE: 5
gtccaagctt gccaccatgg attatcaagt gtca       
#                  
#        34
<210> SEQ ID NO 6
<211> LENGTH: 61
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide
<400> SEQUENCE: 6
ctagctcgag tcaagcgtag tctgggacgt cgtatgggta gcacaagccc ac
#agatattt     60
c                  
#                  
#                  
#               61
<210> SEQ ID NO 7
<211> LENGTH: 30
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide
<400> SEQUENCE: 7
cgggatccct ccatggatta tcaagtgtca         
#                  
#           30
<210> SEQ ID NO 8
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide
<400> SEQUENCE: 8
cgggatcccg ctcacaagcc cacagatat         
#                  
#            29
<210> SEQ ID NO 9
<211> LENGTH: 329
<212> TYPE: PRT
<213> ORGANISM: Protein
<400> SEQUENCE: 9
Glu Glu Val Thr Thr Phe Phe Asp Tyr Asp Ty
#r Gly Ala Pro Cys His
1               5   
#                10  
#                15
Lys Phe Asp Val Lys Gln Ile Gly Ala Gln Le
#u Leu Pro Pro Leu Tyr
            20      
#            25      
#            30
Ser Leu Val Phe Ile Phe Gly Phe Val Gly As
#n Met Leu Val Val Leu
        35          
#        40          
#        45
Ile Leu Ile Asn Cys Lys Lys Leu Lys Cys Le
#u Thr Asp Ile Tyr Leu
    50              
#    55              
#    60
Leu Asn Leu Ala Ile Ser Asp Leu Leu Phe Le
#u Ile Thr Leu Pro Leu
65                  
#70                  
#75                  
#80
Trp Ala His Ser Ala Ala Asn Glu Trp Val Ph
#e Gly Asn Ala Met Cys
                85  
#                90  
#                95
Lys Leu Phe Thr Gly Leu Tyr His Ile Arg Ty
#r Leu Ala Ile Val His
            100      
#           105      
#           110
Ala Val Phe Ala Leu Lys Ala Arg Thr Val Th
#r Phe Gly Val Val Thr
        115          
#       120          
#       125
Ser Val Ile Thr Trp Leu Val Ala Val Phe Al
#a Ser Val Pro Gly Ile
    130              
#   135              
#   140
Ile Phe Thr Lys Cys Gln Lys Glu Asp Ser Va
#l Tyr Val Cys Gly Pro
145                 1
#50                 1
#55                 1
#60
Tyr Phe Pro Arg Gly Trp Asn Asn Phe His Th
#r Ile Met Arg Asn Ile
                165  
#               170  
#               175
Leu Gly Leu Val Leu Pro Leu Leu Ile Met Va
#l Ile Cys Tyr Ser Gly
            180      
#           185      
#           190
Ile Leu Lys Thr Leu Leu Arg Cys Arg Asn Gl
#u Lys Lys Arg His Arg
        195          
#       200          
#       205
Ala Val Arg Val Ile Phe Thr Ile Met Ile Va
#l Tyr Phe Leu Phe Trp
    210              
#   215              
#   220
Thr Pro Tyr Asn Ile Val Ile Leu Leu Asn Th
#r Phe Gln Glu Phe Phe
225                 2
#30                 2
#35                 2
#40
Gly Leu Ser Asn Cys Glu Ser Thr Ser Gln Le
#u Asp Gln Ala Thr Gln
                245  
#               250  
#               255
Val Thr Glu Thr Leu Gly Met Thr His Cys Cy
#s Ile Asn Pro Ile Ile
            260      
#           265      
#           270
Tyr Ala Phe Val Gly Glu Lys Phe Arg Ser Le
#u Phe His Ile Ala Leu
        275          
#       280          
#       285
Gly Cys Arg Ile Ala Pro Leu Gln Lys Pro Va
#l Cys Gly Gly Pro Gly
    290              
#   295              
#   300
Val Arg Pro Gly Lys Asn Val Lys Val Thr Th
#r Gln Gly Leu Leu Asp
305                 3
#10                 3
#15                 3
#20
Gly Arg Gly Lys Gly Lys Ser Ile Gly
                325

Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US5314992Nov 25, 1991May 24, 1994Trustees Of Dartmouth CollegeLipocortin-1 receptor protein and its uses
US5440021Feb 25, 1994Aug 8, 1995Chuntharapai; AnanAntibodies to human IL-8 type B receptor
US5652133Jan 28, 1993Jul 29, 1997The United States Of America As Represented By The Secretary Of The Department Of Health And Human ServicesCloning and expression of the human macrophage inflammatory protein-1.alpha.α) /rantes receptor
US5707815May 25, 1995Jan 13, 1998Regents Of The University Of CaliforniaMammalian monocyte chemoattractant protein receptors and assays using them
US5798206Jan 10, 1997Aug 25, 1998New York Blood CenterMethods for screening of test compounds for inhibiting binding of a CD4-HIV 1 complex to a chemokine receptor
US5817310Jun 10, 1994Oct 6, 1998Cor Therapeutics, Inc.Inhibitory immunoglobulin polypeptides to human PDGF beta receptor
US5912176Jun 2, 1997Jun 15, 1999United Biomedical, Inc.Antibodies against a host cell antigen complex for pre and post exposure protection from infection by HIV
US5919776Dec 18, 1997Jul 6, 1999Merck & Co., Inc.Substituted aminoquinolines as modulators of chemokine receptor activity
US5928881Jul 11, 1997Jul 27, 1999Smithkline Beecham CorporationMethod of identifying agonists and antagonist for CC-CKR5 receptor
US5939320Jun 19, 1996Aug 17, 1999New York UniversityG-coupled receptors associated with macrophage-trophic HIV, and diagnostic and therapeutic uses thereof
US5939538Dec 19, 1996Aug 17, 1999Immusol IncorporatedMethods and compositions for inhibiting HIV infection of cells by cleaving HIV co-receptor RNA
US5961976Feb 28, 1997Oct 5, 1999United Biomedical, Inc.Antibodies against a host cell antigen complex for pre- and post-exposure protection from infection by HIV
US5962462Dec 12, 1997Oct 5, 1999Merck & Co., Inc.Spiro-substituted azacycles as modulators of chemokine receptor activity
US5994515Jun 25, 1997Nov 30, 1999Trustees Of The University Of PennsylvaniaAntibodies directed against cellular coreceptors for human immunodeficiency virus and methods of using the same
US6013644Dec 12, 1997Jan 11, 2000Merck & Co., Inc.Spiro-substituted azacycles as modulators of chemokine receptor activity
US6025154Jun 6, 1995Feb 15, 2000Human Genome Sciences, Inc.Polynucleotides encoding human G-protein chemokine receptor HDGNR10
US6057102Aug 8, 1997May 2, 2000The Aaron Diamond Aids Research CenterHIV coreceptor mutants
US6132987 *Jan 11, 1995Oct 17, 2000The Regents Of The University Of CaliforniaRecombinant mammalian monocyte chemotactic protein-1 (MCP-1) receptors (MCP-1R, CCR-2)
US6153431 *May 28, 1998Nov 28, 2000Fond Mondiale Rech & Prev SidaHuman immunodeficiency virus co-receptor variants associated with resistance to virus infection
US6258527 *May 21, 1997Jul 10, 2001The Aaron Diamond Aids Research CenterMethods of identifying g-coupled receptors associated with macrophage-trophic HIV, and diagnostic and therapeutic uses thereof
US6265184 *Dec 20, 1995Jul 24, 2001Icos CorporationPolynucleotides encoding chemokine receptor 88C
US6268477Jun 7, 1996Jul 31, 2001Icos CorporationChemokine receptor 88-C
US6287805 *Mar 20, 1998Sep 11, 2001Millennium Pharmaceuticals, Inc.Nucleic acid molecules of the protein-coupled heptahelical receptor superfamily and uses therefor
CA2128208A1Nov 17, 1993Jun 9, 1994Icos CorpNovel seven transmembrane receptors
CA2146328A1Nov 4, 1993May 26, 1994Genentech, Inc.C-c ckr-1, c-c chemokine receptor
EP0612723A1Feb 16, 1994Aug 31, 1994Hoechst AktiengesellschaftSubstituted benzoylguanidines, process for their preparation, their use as medicament, as inhibitor of the cellular Na+/H+ exchange or as diagnostic agent and medicament containing them
EP0671391A1Feb 27, 1995Sep 13, 1995Bayer Ag5-Vinyl- and 5-ethinyl-quinolinone- and -naphthyridone-carboxylic acids
EP0834564A2Oct 3, 1997Apr 8, 1998Smithkline Beecham CorporationA mouse genomic clone of the CC-CKR5 receptor
EP0979655A1Aug 1, 1998Feb 16, 2000Boehringer Mannheim GmbhMeans for treatment of infections caused by RNA-Virusses such as HIV
FR2771423A1 Title not available
JPH10179180A Title not available
JPH11243960A Title not available
JPH11292795A Title not available
RU2126048C1 Title not available
WO1994011504A1Nov 4, 1993May 26, 1994Genentech IncC-c ckr-1, c-c chemokine receptor
WO1995019436A1Jan 11, 1995Jul 20, 1995Israel CharoMammalian monocyte chemoattractant protein receptors
WO1996022371A2Jan 19, 1996Jul 25, 1996Brigham & Womens HospitalC-C CHEMOKINE RECEPTOR 3: CKP-3 OR Eos-L2
WO1996023068A1Jan 24, 1996Aug 1, 1996Glaxo Group LtdA chemokine receptor able to bind to mcp-1, mip-1 alpha and/or rantes. its uses
WO1996038559A1May 22, 1996Dec 5, 1996Dana Farber Cancer Inst IncChemokine n-terminal deletion mutations
WO1996039437A1Jun 6, 1995Dec 12, 1996Human Genome Sciences IncHuman g-protein chemokine receptor hdgnr10
WO1997000960A1Jun 21, 1996Jan 9, 1997Leukosite IncNovel human chemotactic cytokine
WO1997019696A1Nov 27, 1996Jun 5, 1997Paolo LussoC-c chemokines that inhibit retrovirus infection
WO1997021812A2Dec 5, 1996Jun 19, 1997Schering CorpMammalian chemokine ccf8 and chemokine receptor cckr3
WO1997022698A2Dec 20, 1996Jun 26, 1997Icos CorpChemokine receptors 88-2b[ckr-3] and 88c and their antibodies
WO1997025340A1Jan 11, 1996Jul 17, 1997Human Genome Sciences IncHuman g-protein chemokine receptor hsatu68
WO1997028258A1Jan 30, 1997Aug 7, 1997Nat Inst HealthCells expressing both human cd4 and cxcr4
WO1997032019A2Feb 28, 1997Sep 4, 1997Euroscreen SaC-c ckr-5, cc-chemikines receptor, derivatives thereof and their uses
WO1997035881A2Mar 26, 1997Oct 2, 1997Dowd Brian F OReceptor and transporter antagonists
WO1997037005A1Apr 2, 1997Oct 9, 1997Progenics Pharm IncMethod for preventing hiv-1 infection of cd4+ cells
WO1997041225A2Apr 25, 1997Nov 6, 1997Au Young JaniceMammalian mixed lymphocyte receptors, chemokine receptors [mmlr-ccr]
WO1997041230A2Apr 30, 1997Nov 6, 1997Forssmann Wolf GeorgCc-type chemokines
WO1997044055A1May 20, 1997Nov 27, 1997Univ New YorkMethods of identifying g-coupled receptors associated with macrophage-trophic hiv, and diagnostic and therapeutic uses thereof
WO1997045543A2May 28, 1997Dec 4, 1997Us HealthCc chemokine receptor 5, antibodies thereto, transgenic animals
WO1997046697A2Jun 3, 1997Dec 11, 1997United Biomedical IncAntibodies against a complex of cd4 and a chemokine receptor domain, and their use against hiv infections
WO1997047318A1Jun 13, 1997Dec 18, 1997Aaron Diamond Aids Research CeUses of a chemokine receptor for inhibiting hiv-1 infection
WO1997047319A1Jun 13, 1997Dec 18, 1997Aaron Diamond Aids Research CeUses of a chemokine receptor for inhibiting hiv-1 infection
WO1997049424A1Jun 25, 1997Dec 31, 1997Univ PennsylvaniaAntibodies directed against cellular coreceptors for human immunodeficiency virus and methods of using the same
WO1998000535A2Jun 27, 1997Jan 8, 1998Dana Farber Cancer Inst IncMethod for inhibiting hiv-1 infection, drug screens, and methods of diagnosis and prognosis of susceptibility to hiv infection
WO1998005798A1Aug 7, 1997Feb 12, 1998Aaron Diamond Aids Research CeHiv coreceptor mutants
WO1998014480A1Sep 24, 1997Apr 9, 1998Leukosite IncG protein-coupled receptor antagonists
WO1998015569A1Oct 8, 1997Apr 16, 1998Dana Farber Cancer Inst IncGp120 polypeptides having conformational discontinuous chemokine receptor binding sites and methods of inhibiting hiv infection
WO1998017308A1Oct 24, 1997Apr 30, 1998Jack BarberInhibiting hiv infection by cleaving co-receptor rna
WO1998018826A2Oct 27, 1997May 7, 1998Leukosite IncAnti-ccr5 antibodies and methods of use therefor
WO1998020166A2Nov 6, 1997May 14, 1998Andreas BraunDna diagnostics based on mass spectrometry
WO1998025604A1Dec 12, 1997Jun 18, 1998Maccoss MalcolmSpiro-substituted azacycles as modulators of chemokine receptor activity
WO1998025605A1Dec 12, 1997Jun 18, 1998Malcolm MaccossSpiro-substituted azacycles as modulators of chemokine receptor activity
WO1998025617A1Dec 12, 1997Jun 18, 1998Malcolm MaccossSubstituted aryl piperazines as modulators of chemokine receptor activity
WO1998027815A1Dec 18, 1997Jul 2, 1998William K HagmannSubstituted aminoquinolines as modulators of chemokine receptor activity
WO1998030218A1Jan 8, 1998Jul 16, 1998William E BondinellSubstituted bis-acridines and related compounds as ccr5 receptor ligands, anti-inflammatory agents and anti-viral agents
WO1998031364A1Jan 20, 1998Jul 23, 1998Timothy Harrison3,3-disubstituted piperidines as modulators of chemokine receptor activity
WO1998034945A1Feb 6, 1998Aug 13, 1998Epoch Pharmaceuticals IncTargeted modification of the ccr-5 gene
WO1998038212A2Feb 27, 1998Sep 3, 1998Genetics InstChimeric polypeptides containing chemokine domains
WO1998042354A1Mar 26, 1998Oct 1, 1998Univ Maryland Biotech InstChemokines that inhibit immunodeficiency virus infection and methods based thereon
WO1998044158A1Mar 27, 1998Oct 8, 1998Epitope IncSimultaneous collection of dna and non-nucleic analytes from oral fluids
WO1998051705A1May 11, 1998Nov 19, 1998Lusso PaoloPeptides with antiviral activity
WO1998054317A1May 29, 1998Dec 3, 1998Fond Mondiale Rech & Prev SidaHuman immunodeficiency virus co-receptor variants associated with resistance to virus infection
WO1998055873A2Jun 4, 1998Dec 10, 1998Philippe AlixUse of a fluorescent protein for detecting interaction between a target protein and its ligand
WO1998056421A1Jun 12, 1998Dec 17, 1998Graham P AllawayMethod for preventing hiv-1 infection of cd4+ cells
WO1998058536A1Jun 22, 1998Dec 30, 1998Israel F CharoTransgenic rabbits expressing cd4 and chemokine receptor
WO1998058966A1Jun 24, 1997Dec 30, 1998Godin NormanA composition for specific immunoprotection and method for obtaining said composition
WO1999001127A1Jul 1, 1998Jan 14, 1999William E BondinellCompounds and methods
WO1999004794A1Jul 21, 1998Feb 4, 1999Charles G CaldwellCyclic amine modulators of chemokine receptor activity
WO1999006561A2Jul 29, 1998Feb 11, 1999Ghalib AlkhatibChemokine receptor ccr8 dna and uses thereof
WO1999008703A1Aug 14, 1998Feb 25, 1999Picower Inst Med ResAnti-viral treatment with pertussis toxin b oligomer
WO1999009984A1Aug 27, 1998Mar 4, 1999Scott C BerkPyrrolidine and piperidine modulators of chemokine receptor activity
WO1999012416A1Sep 9, 1998Mar 18, 1999Univ ColumbiaT-independent conjugate-vaccines
WO1999013112A1Sep 14, 1998Mar 18, 1999Akzo Nobel NvCcr5 rna transcription based amplification assay
WO1999014378A2Sep 18, 1998Mar 25, 1999Pavlakis George NMethods using interleukin 4 to treat hiv infection
WO1999017773A1Oct 7, 1998Apr 15, 1999William E BondinellCompounds and methods
WO1999023107A1Oct 30, 1998May 14, 1999Maxygen IncModification of virus tropism and host range by viral genome shuffling
WO1999023253A1Oct 23, 1998May 14, 1999Brien Stephen J OStromal cell derived factor-1 (sdf-1) and method of use for diagnostic and prognostic indicator of aids pathogenesis
WO1999024065A1Nov 10, 1998May 20, 1999Dana Farber Cancer Inst IncCOMPOUNDS INHIBITING CD4-gp120 INTERACTION AND USES THEREOF
WO1999024553A2Nov 10, 1998May 20, 1999Univ ColumbiaX-ray crystal comprising hiv-1 gp120
WO1999027122A1Nov 23, 1998Jun 3, 1999Transgene SaVectors inhibiting or delaying the binding of an immunodeficiency virus to cells
WO1999027939A1Dec 4, 1998Jun 10, 1999O M Zack HowardUreido derivatives of poly-4-amino-2-carboxy-1-methyl pyrrole compounds for inhibition of inflammation
WO1999028474A2Dec 1, 1998Jun 10, 1999Michael A NorcrossChemokine variants and methods of use
WO1999032100A2Dec 17, 1998Jul 1, 1999Takeda Chemical Industries LtdPharmaceutical composition for antagonizing ccr5 comprising anilide derivative
WO1999032138A1Dec 18, 1998Jul 1, 1999Immunex CorpMethod for reducing susceptibility to hiv infection
WO1999033989A2Dec 21, 1998Jul 8, 1999Paolo LussoRantes mutants and therapeutic applications thereof
WO1999036518A1Jan 15, 1999Jul 22, 1999Btg Int LtdRibozymal nucleic acids cleaving ccr5 or cxcr4
WO1999038514A1Feb 1, 1999Aug 5, 1999Charles G CaldwellCyclic amine modulators of chemokine receptor activity
WO1999043711A1Feb 26, 1999Sep 2, 1999Christopher J MichejdaG protein-coupled receptor antagonists
WO1999046372A2Mar 5, 1999Sep 16, 1999Hope CityRibozymes capable of inhibiting the expression of the ccr5 receptor
WO1999051751A1Apr 1, 1999Oct 14, 1999Toru KimuraHiv cofactor inhibitors and medicinal compositions for preventing or treating hiv-infection
Non-Patent Citations
Reference
1"Myocardial Infarction," in The Merck Manual of Diagnosis and Therapy, Seventeenth Edition, Centennial Edition, Section 16, Chapter 202, Beers, M.H. and Berkow, R., eds., Merck & Co., Inc. (1999) <http://www.merck.com/pubs/Manual/section16/chapter202/202d.htm> (visited Aug. 2001).
2Alkhatib, G. et al., "CC CKR5: A RANTES, MIP-1alpha, MIP-1beta Receptor as a Fusion Cofactor for Macrophage-Tropic HIV-1," Science 272:1955-1958, American Association for the Advancement of Science (Jun. 1996).
3Alkhatib, G. et al., "CC CKR5: A RANTES, MIP-1α, MIP-1β Receptor as a Fusion Cofactor for Macrophage—Tropic HIV-1," Science 272:1955-1958, American Association for the Advancement of Science (Jun. 1996).
4Balter, M., "A Second Coreceptor for HIV In Early Stages of Infection," Science 272:1740, American Association for the Advancement of Science (Jun. 1996).
5Balter, M., "Elusive HIV-Suppressor Factors Found," Science 270:1560-1561, American Association for the Advancement of Science (Dec. 1995).
6Bendig, M.M., "Humanization of Rodent Monoclonal Antibodies by CDR Grafting," Methods 8:83-93 (Oct. 1995) (Academic Press, Inc.).
7Bowie, J.U., et al. "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (Mar. 1990) (American Association for the Advancement of Science).
8Brühl, H. et al., "Depletion of CCR5-Expressing Cells with Bispecific Antibodies and Chemokine Toxins: A New Strategy in the Treatment of Chronic Inflammatory Diseases and HIV," J. Immunol. 166:2420-2426, The American Association of Immunologists (Feb. 2001).
9Charo, I.F. et al., "Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails," Proc. Natl. Acad. Sci. USA 91:2752-2756 (1994).
10Choe, H. et al., "The beta-Chemokine Receptors CCR3 and CCR5 Facilitate Infection by Primary HIV-1 Isolates," Cell 85:1135-1148, Cell Press (Jun. 1996).
11Choe, H. et al., "The β-Chemokine Receptors CCR3 and CCR5 Facilitate Infection by Primary HIV-1 Isolates," Cell 85:1135-1148, Cell Press (Jun. 1996).
12Cocchi, F. et al., "Identification of RANTES, MIP-1alpha, and MIP-1beta as the Major HIV-Suppressive Factors Produced by CD8+ T Cells," Science 270:1811-1815, American Association for the Advancement of Science (Dec. 1995).
13Cocchi, F. et al., "Identification of RANTES, MIP-1α, and MIP-1β as the Major HIV-Suppressive Factors Produced by CD8+ T Cells," Science 270:1811-1815, American Association for the Advancement of Science (Dec. 1995).
14Cohen, J., "Likely HIV Cofactor Found," Science 272:809-810, American Association for the Advancement of Science (May 1996).
15Combadiere, C. et al., "Additions and Corrections to: Cloning and functional expression of a human eosinophil CC chemokine receptor," J. Biol. Chem. 270:30235, The American Society for Biochemistry and Molecular Biology (Dec. 1995).
16Combadiere, C. et al., "Cloning and functional expression of a human eosinophil CC chemokine receptor," J. Biol. Chem. 270:16491-16494, American Society for Biochemistry and Molecular Biology (Jul. 1995).
17Combadiere, C. et al., "Cloning and functional expression of CC CKR5, a human monocyte CC chemokine receptor selective for MIP-1alpha, MIP-1beta, and RANTES," J. Leukocyte Biol. 60:147-152, The Society for Leukocyte Biology (Jul. 1996).
18Combadiere, C. et al., "Monocyte Chemoattractant Protein-3 Is a Functional Ligand for CC Chemokine Receptors 1 and 2," J. Biol. Chem. 270:29671-29675, American Society for Biochemistry and Molecular Biology (Dec. 1995).
19Combadiere, C. et al., "Cloning and functional expression of CC CKR5, a human monocyte CC chemokine receptor selective for MIP-1α, MIP-1β, and RANTES," J. Leukocyte Biol. 60:147-152, The Society for Leukocyte Biology (Jul. 1996).
20Cunningham, M.W., et al., "Cytotoxic and Viral Neutralizing Antibodies Crossreact With Streptococcal M Protein, Enteroviruses, and Human Cardiac Myosin," Proc. Natl. Acad. Sci. USA 89:1320-1324 (Feb. 1992) (National Academy of Sciences).
21Deng, H. et al., "Identification of a major co-receptor for primary isolates of HIV-1," Nature 381:661-666, Macmillan Publishing Group (Jun. 1996).
22Dialog, File 351 (Derwent), English language abstract of EP 0 612 723.
23Dialog, File 351 (Derwent), English language abstract of EP 0 671 391.
24Dialog, File 351 (Derwent), English language abstract of EP 0 979 655.
25Dialog, File 351 (Derwent), English language abstract of FR 2 771 423.
26Dialog, File 351 (Derwent), English language abstract of JP 11-243960.
27Dialog, File 351 (Derwent), English language abstract of JP 11-292795.
28Dialog, File 351 (Derwent), English language abstract of RU 2126048.
29Dimitrov, D.S., "Fusin-a place for HIV-1 and T4 cells to meet," Nature Med. 2:640-641, Nature Publishing Group (Jun. 1996).
30Dimitrov, D.S., "Fusin—a place for HIV-1 and T4 cells to meet," Nature Med. 2:640-641, Nature Publishing Group (Jun. 1996).
31Doranz, B.J. et al., "A Dual-Tropic Primary HIV-1 Isolate That Uses Fusin and the beta-Chemkine Receptors CKR-5, CKR-3, and CKR-2b as Fusion Cofactors," Cell 85:1149-1158, Cell Press (Jun. 1996).
32Doranz, B.J. et al., "A Dual-Tropic Primary HIV-1 Isolate That Uses Fusin and the β-Chemkine Receptors CKR-5, CKR-3, and CKR-2b as Fusion Cofactors," Cell 85:1149-1158, Cell Press (Jun. 1996).
33Dragic, T. et al., "HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5," Nature 381:667-673, Macmillan Publishing Group (Jun. 1996).
34Eva, C. et al., "Molecular cloning of a novel G protein-coupled receptor that may belong to the neuropeptide receptor family," FEBS Letters 271:81-84 (1990).
35Feng, Y. et al., "HIV-1 Entry Cofactor: Functional cDNA Cloning of a Seven-Transmembrane, G Protein-Coupled Receptor," Science 272:872-877, America Association for the Advancement of Science (May 1996).
36Gao, J.-L. et al., "Structure and Functional Expression of the Human Macrophage Inflammatory Protein 1alpha/RANTES Receptor," J. Exp. Med. 117:1421-1427 (1993).
37Gao, J.-L. et al., "Structure and Functional Expression of the Human Macrophage Inflammatory Protein 1α/RANTES Receptor," J. Exp. Med. 117:1421-1427 (1993).
38GenBank Report, Accession No. AF019772, submitted by Doranz, B.J. et al. (Sep. 1997).
39George, D.G. et al., "Chapter 12. Current Methods in Sequence Comparison and Analysis," in: Macromolecular Sequencing and Synthesis. Selected Methods and Applications, Alan R. Liss, Inc., pp. 127-149 (1988).
40Gomez-Reino, J.J. et al., "Association of Rheumatoid Arthritis with a Functional Chemokine Receptor, CCR5," Arthritis Rheum. 42:989-992, American College of Rheumatology (May 1999).
41Gura, T., "Chemokines Take Center Stage in Inflammatory Ills," Science 272:954-956, American Association for the Advancement of Science (May 1996).
42Hla, T. and T. Maciag, "An abundant transcript induced in differentiating human endothelial cells encodes a polypeptide with structural similarities to G-protein-coupled receptors," J. Biol. Chem. 265:9308-9313 (1990).
43Horuk, R., "Molecular Properties of the Chemokine Receptor Family," Trends in Pharmacol. Sci. 15:159-165 (May 1994) (Elsevier Science).
44Jobling, M.G. and Holmes, R.K., "Analysis of structure and function of the B subunit of cholera toxin by the use of site-directed mutagenesis," Mol. Microbiol. 5:1755-1767, Blackwell Science (1991).
45Lee, B., et al., "Epitope Mapping of CCR5 Reveals Multiple Conformational States and Distinct but Overlapping Structures Involved in Chemokine and Coreceptor Function," J. Biol. Chem. 274:9617-9626 (Apr. 1999) (American Society for Biochemistry and Molecular Biology).
46Lee, N. and Kerlavage, A.R., "Molecular Biology of G-Protein-Coupled Receptors," Trends in Biomedical Research 6: 488-497 (1993).
47Lee, N.H. and Kerlavage, A.R., "Molecular Biology of G-Protein-Coupled Receptors," DN & P 6:488-497, Prous Science (1993).
48Leong, S.R., et al., "Complete Mutagenesis of the Extracellular Domain of Interleukin-8 (IL-8) Type A Receptor Identifies Charged Residues Mediating IL-8 Binding and Signal Transduction," J. Biol. Chem. 269:19343-19348 (Jul. 1994) (American Society for Biochemistry and Molecular Biology).
49Lerner, R.A., "Tapping the immunological repertoire to produce antibodies of predetermined specificity," Nature 299:592-596, Macmillan Journals Ltd (1982).
50Libert, F. et al., "Selective amplification and cloning of four new members of the G protein-coupled receptor family," Science 244:569-572 (1989).
51Mack, M. et al., "Predominance of Mononuclear Cells Expressing the Chemokine Receptor CCR5 in Synovial Effusions of Patients with Different Forms of Arthritis," Arthritis Rheum. 42:981-988, American College of Rheumatology (May 1999).
52Marshall, E., "HIV Experts vs. Sequencers in Patent Race," Science 275:1263, American Association for the Advancement of Science (Feb. 1997).
53Meyerhof, W. et al., "Molecular cloning of a novel putative G-protein coupled receptor expressed during rat spermiogenesis," FEBS Letters 284:155-160 (1991).
54Murdoch, C. and Finn, A., "Chemokine receptors and their role in inflammation and infectious diseases," Blood 95:3032-3043, The American Society of Hematology (May 2000).
55Murphy, P.M., "The Molecular Biology of Leukocyte Chemoattractant Receptors," Annu. Rev. Immunol. 12:593-633, Annual Reviews, Inc. (1994).
56NCBI Entrez, Genbank Report, Accession No. D10925, Nomura, H. et al. (Sep. 1994).
57NCBI Entrez, Genbank Report, Accession No. D29984, Yamagami, S. et al. (1996).
58NCBI Entrez, Genbank Report, Accession No. L09230, Neote, K. et al. (Dec. 1994).
59NCBI Entrez, Genbank Report, Accession No. L10918, Gao, J.-L. et al. (1993).
60NCBI Entrez, Genbank Report, Accession No. L24445, Prado, G.N. et al. (May 1994).
61NCBI Entrez, Genbank Report, Accession No. M74240, Beckman, M.P. et al. (1991).
62NCBI Entrez, Genbank Report, Accession No. U03882, Charo, I.F. et al. (Jun. 1994).
63NCBI Entrez, Genbank Report, Accession No. U28406, Gao, J.L. and Murphy, P.M. (1996).
64NCBI Entrez, Genbank Report, Accession No. U28694, Combadiere, C. et al. (1996).
65NCBI Entrez, Genbank Report, Accession No. U29677, Post, T.W. et al. (1996).
66NCBI Entrez, Genbank Report, Accession No. U47035, Boring, L. et al. (1996).
67NCBI Entrez, Genbank Report, Accession No. U47036, Boring, L. et al. (1996).
68NCBI Entrez, Genbank Report, Accession No. U49727, Ponath, P.D. et al. (1996).
69NCBI Entrez, Genbank Report, Accession No. U51241, Daugherty, B.L. (1996).
70NCBI Entrez, Genbank Report, Accession No. U51717, Kurihara, T. and Bravo, R. (1996).
71NCBI Entrez, Genbank Report, Accession No. U54994, Raport, C.J. et al. (1996).
72NCBI Entrez, Genbank Report, Accession No. U56819, Heesen, M. et al. (1996).
73NCBI Entrez, Genbank Report, Accession No. U57840, Combadiere, C. et al. (1996).
74NCBI Entrez, Genbank Report, Accession No. U68565, Kuziel, W.A. and Maeda, N. (1996).
75NCBI Entrez, Genbank Report, Accession No. U70988, Dunstan, C.A.N. et al. (1996).
76NCBI Entrez, Genbank Report, Accession No. X91492, Samson, M. et al. (1996).
77NCBI Entrez, Genbank Report, Accession No. X94151, Meyer, A. et al. (1996).
78NCBI Entrez, Genbank Report, Accession No. X99393, Samson, M. et al. (1996).
79Neote, K. et al., "Molecular Cloning, Functional Expression, and Signaling Characteristics of a C-C Chemokine Receptor," Cell 72:415-425 (1993).
80Nomura, H. et al., "Molecular cloning of cDNAs encoding a LD78 receptor and putative leukocyte chemotactic peptide receptors," Intl. Immunol. 5:1239-1249 (1993).
81Oliveira, L. et al., "A common motif in G-protein-coupled seven transmembrane helix receptors," J. Comput. Aided Mol. Des. 7: 649-658 (1993).
82Olson, W., et al., "Differential Inhibition of Human Immunodeficiency Virus Type 1 Fusion, gp120 Binding, and CC-Chemokine Activity by Monoclonal Antibodies to CCR5," J. Virol. 73:4145-4155 (May 1999) (American Society for Microbiology).
83Osbourn, J.K., et al., "Directed Selection of MIP-Ialpha Neutralizing CCR5 Antibodies from a Phage Display Human Antibody Library," Nature Biotechnol. 16:778-781 (Aug. 1998) (Nature Publishing Group).
84Osbourn, J.K., et al., "Directed Selection of MIP-Iα Neutralizing CCR5 Antibodies from a Phage Display Human Antibody Library," Nature Biotechnol. 16:778-781 (Aug. 1998) (Nature Publishing Group).
85Probst, W.C. et al., "Sequence Alignment of the G-Protein Coupled Receptor Superfamily," DNA and Cell Biology 11: 1-20 (1992).
86 *Promega Catalog 1993-1994 3: random primers.*
87Raport, C.J. et al., "Molecular Cloning and Functional Characterization of a Novel Human CC Chemokine Receptor (CCR5) for RANTES, MIP-1beta, and MIP-1alpha," J. Biol. Chem. 271:17161-17166, American Society for Biochemistry and Molecular Bilogy (Jul. 1996).
88Raport, C.J. et al., "Molecular Cloning and Functional Characterization of a Novel Human CC Chemokine Receptor (CCR5) for RANTES, MIP-1β, and MIP-1α," J. Biol. Chem. 271:17161-17166, American Society for Biochemistry and Molecular Bilogy (Jul. 1996).
89Ross, P.C. et al., "RTA, a candidate G protein-coupled receptor: cloning, sequencing, and tissue distribution," Proc. Natl. Acad. Sci. USA 87:3052-3056 (1990).
90Samson, M. et al., "Molecular Cloning and Functional Expression of a New Human CC-Chemokine Receptor Gene," Biochemistry 35:3362-3367, American Chemical Society (Mar. 1996).
91Samson, M. et al., "Molecular Cloning and Functional Expression of a New Human CC-Chemokine Receptor Gene," Chem. Abstracts 124:993, Abstract No. 258056e, The American Chemical Society (May 1996).
92Schall, T.J., "Biology of the RANTES/SIS Cytokine Family," Cytokine 3:165-183 (May 1991) (Academic Press, Inc.).
93Skolnick, J. and Fetrow, J.S., "From genes to protein structure and function: novel applications of computational approaches in the genomic era," TIBTECH 18:34-39, Elsevier Science Ltd (Jan. 2000).
94Suzuki, N. et al., "Selective accumulation of CCR5+ T lymphocytes into inflamed joints of rheumatoid arthritis," Intl. Immunol. 11:553-559, The Japanese Society for Immunology and Oxford University Press (Apr. 1999).
95Travis, J., "Multiple doors for HIV to enter cells," Science News 149:390, Science Service (Jun. 1996).
96Weiss, R.A. and P.R. Clapham, "Hot fusion of HIV," Nature 381:647-648, Macmillan Publishing Group (Jun. 1996).
97Wu, L., et al., "Interaction of Chemokine Receptor CCR5 with its Ligands: Multiple Domains for HIV-1 gp 120 Binding and a Single Domain for Chemokine Binding," J. Exp. Med. 186:1373-1381 (Oct. 1997) (The Rockefeller University Press).
98Yamagami, S. et al., "cDNA Cloning and Functional Expression of a Human Monocyte Chemoattractant Protein 1 Receptor," Biochem. Biophys. Res. Comm. 202:1156-1162 (1994).
99Zang, Y.C.Q. et al., "Aberrant T cell migration toward RANTES and MIP-1alpha in patients with multiple sclerosis: Overexpression of chemokine receptor CCR5," Brain 123:1874-1882, Oxford University Press (Sep. 2000).
100Zang, Y.C.Q. et al., "Aberrant T cell migration toward RANTES and MIP-1α in patients with multiple sclerosis: Overexpression of chemokine receptor CCR5," Brain 123:1874-1882, Oxford University Press (Sep. 2000).
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Classifications
U.S. Classification435/69.1, 435/320.1, 435/252.3, 536/23.1
International ClassificationC12P21/08, C07K14/715, A61K38/00
Cooperative ClassificationC07K14/7158, A61K38/00, C12N2799/026
European ClassificationC07K14/715H
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